Effects of Mood Inductions by Meal Ambiance and Moderate Alcohol Consumption on Endocannabinoids and N-Acylethanolamines in Humans: A Randomized Crossover Trial

Background The endocannabinoid system is suggested to play a regulatory role in mood. However, the response of circulating endocannabinoids (ECs) to mood changes has never been tested in humans. In the present study, we examined the effects of mood changes induced by ambiance and moderate alcohol consumption on plasma ECs 2-arachidonoylglycerol (2-AG), anandamide (AEA), and some N-acylethanolamine (NAE) congeners in humans. Methods Healthy women (n = 28) participated in a randomized cross-over study. They consumed sparkling white wine (340 mL; 30 g alcohol) or alcohol-free sparkling white wine (340 mL; <2 g alcohol) as part of a standard evening meal in a room with either a pleasant or an unpleasant ambiance. Results Plasma concentrations of palmitoylethanolamide (PEA) and stearoylethanolamide (SEA) increased after 30 min in the unpleasant ambiance, while they decreased in the pleasant ambiance. Changes in ECs and their NAE congeners correlated with mood states, such as happiness and fatigue, but in the pleasant ambiance without alcohol only. ECs and their NAE congeners were correlated with serum free fatty acids and cortisol. Conclusion This is the first human study to demonstrate that plasma NAEs are responsive to an unpleasant meal ambiance. Furthermore, associations between mood states and ECs and their NAE congeners were observed. Trial Registration Clinicaltrials.gov NCT01426022


Facility for the preparation and dispense of pharmaceutical products
Not applicable

Responsibilities
The sponsor will be responsible for the financial compensation for the conduct of the study. Moreover the sponsor is responsible for the insurance according to the "WMO". Insurances for material damage and accidents during the travel to and from TNO and during the stay at TNO are TNO's responsibility.
Mr Hendriks will be responsible for the overall conduct of the study. Ms Schrieks assists in the daily conduct of the study. Mr Hendriks will also be responsible for monitoring dietary intake, receipt, coding, distribution, storage, accountability, and labelling of study substances. Most of these actions will be carried out by Mrs Fick-Brinkhof.
Mrs Klöpping-Ketelaars will be responsible for the selection of the subjects, medical aspects of the study, documentation, and interpretation and reporting of the AEs and SAEs.
Mrs Fick-Brinkhof will be responsible for the daily conduct of the clinical part of the study, dispensing of the study substances to the subjects and contacts with the subjects.
Mrs Dutman will be responsible for data management.
Mrs de Jong-Rubingh will be responsible for allocation of the subjects to entry-numbers and treatment.
Mr Gielen will be responsible for measurements of ethanol, insulin, cortisol, glucose and FFA levels.
Mrs van Lipzig will be responsible for measurements of CCK-8, ghrelin, GLP-1 and ACTH.
Mr Schaik will be responsible for measurements of Trp:LNAA ratio.
Mr Coulier will be responsible for measurements of endocannabinoids and N-acylserotonins.
Mr de Groot, Research manager Pharmacokinetics and Human studies, by signing the protocol, declares that the expertise, competence and experience of the local researchers, and the local facilities are appropriate for the adequate conduct of the study, which is consistent with the institution's policies on research and human subjects. Subjects Act) Definitions of terms 'Participants' or 'candidates' refer to the persons who are interested in participating in the study. 'Subjects and volunteers' refer to the persons who actually participated in the study. 'Patients' is not applicable in this study, for this study is performed with apparently healthy subjects.

Summary
Rationale: Food choice is influenced by postprandial mood; the feelings of well-being after a meal. Postprandial mood can be measured by subjective responses. Physiological responses may play an important role in the generation of postprandial mood. However, the relationship between subjective and physiological responses after a meal is not clear yet. To investigate this relationship, moderate alcohol consumption will be used as a mood modulator, because of its well-studied effects on mood. Postprandial mood depends on the current mood state. Therefore we will manipulate the current mood state by changing the ambiance to measure the influence of moderate alcohol consumption with a meal on postprandial mood in a pleasant or unpleasant ambiance.

Primary objective
To determine whether moderate alcohol consumption with a meal in different ambiances affects postprandial mood, evaluated by subjective (POMS, B-BAES, PPW questionnaires) and physiological (ACTH, cortisol, TRP:LNAA ratio, ghrelin blood concentration) parameters.

Secondary objective
To investigate whether moderate alcohol consumption with a meal in different ambiances affects other physiological parameters related to mood, which are not part of the primary objective (endocannabinoids, N-acyl serotonins, β-endorphin, dopamine, insulin, CCK, GLP-1, FFA, glucose, heart rate, HRV and SCL).
Study design: Randomized, placebo-controlled, single-blind, cross-over trial Blinding: Subjects will not be informed about the study objective, but about a study aim that is slightly different. The alcohol content of the drinks will be blinded to the subjects, they will be informed that alcohol content may vary each treatment day. Nature and extent of the burden and risks associated with participation, benefit and group relatedness: Subjects need to visit the study site five times, once for a screening and four times for a treatment day. During these visits blood will be collected five times. The total amount collected during the whole study will be less than 460 mL P9334 | Draft | September 9 th , 2011 11 / 49 TNO Protocol Confidential blood. The study will be performed in women, because women are suspected to have a different postprandial mood response than men (1,2), which might be due to female hormones (3). We will only include women taking oral contraceptives, because they have a reduced variation in female hormones over the menstrual cycle. A large number of women use oral contraceptives nowadays, therefore this group will reflect a large population of women. Women above 45 years will be excluded, because we will include only premenopausal women. Introduction Developing a method to measure and model postprandial mood Food consumption influences our mood, the short-term general feelings of well-being. Previous studies showed that postprandial mood is an important factor in food choice (4,5) and there has been considerable interest in the effects of meals and beverages on postprandial mood. Postprandial mood can be defined as the general feelings of wellbeing after consumption and can be measured by self-reports of subjective feelings in questionnaires. Physiological responses to food intake, such as changes in hormone and neurotransmitter concentrations in the blood, play an important role in the generation of postprandial mood (6). However, the relationship between physiological and subjective responses of postprandial mood is not clear yet. Therefore, the ultimate objective of this study is to develop a method to evaluate postprandial mood and to develop a multi-factorial model describing the associations between physiological postprandial responses and subjective feelings of postprandial mood.
The effect of a food product on postprandial mood, depends also on the current mood state and can either improve, worsen, reinforce or attenuate it. Meal settings differing in environmental factors (e.g. lightening, music, decoration) are known to affect food intake and eating behaviour (7)(8)(9). It is hypothesized that mood plays an important role in these effects (7). Consequently, meal settings differing in ambiance pleasantness are hypothesized to influence the mood state during and after the meal.

Alcohol as mood modulator
To investigate the association between subjective and physiological responses of postprandial mood, moderate alcohol consumption (3 glasses of sparkling white wine; 30g alcohol) will be used as a mood modulator. Extensive research has been done on the effects of moderate alcohol consumption on mood. During rising BACs, alcohol has a stimulating effect, reported by feelings of euphoria, talkativeness and elation. During falling BACs, tension-reducing effects and feelings of fatigue are examples of reported sedative effects (10)(11)(12)(13). The effect of alcohol on mood is suggested to be reinforcing, with greater positive or stimulating effects in a neutral or positive pre-drinking mood and greater sedating effects in a depressive pre-drinking mood (12). Though, the tension-reducing effect of alcohol is an improvement of a negative pre-drinking mood. Non-pharmacological factors (e.g. expectancy and social context) are determining a large part of the alcohol-induced mood change. The expectancy effect is predicted to be as important as the pharmacological effect of alcohol (14). However, Pliner and Cappell (1974) determined the effect of alcohol on mood consumed in different social contexts (drinking solitary or in a group) including a placebo-effect in their model, and concluded that mood changes were even more influenced by social context than by expectancy. They found larger mood changes in individuals drinking in a group compared to drinking alone, and also the type of mood changes was different (15). These findings emphasize that non-pharmacological factors as expectancy and social context are important to take into account when measuring alcohol-induced mood changes.
Although alcohol consumption with dinner is very common in Western countries, only a few studies investigated the influence of alcohol consumption with a meal on postprandial mood (16,17). The combination of food and alcohol consumption may result in a different mood change compared to single alcohol consumption, because food may both directly and indirectly, by influencing alcohol absorption, affect the alcohol-induced mood change (18)(19)(20). Therefore, the aim of this study is to determine the effects of moderate alcohol consumption with a meal in different ambiances on postprandial mood, evaluated by subjective and physiological responses. Thus, the current mood state will be manipulated by environmental factors to induce a pleasant or unpleasant meal ambiance and mood state. Thereby, we will be able to measure the influence of moderate alcohol consumption with a meal on postprandial mood in a positive or negative ambiance.

Subjective response
There are two validated and reliable questionnaires mainly used in previous research to assess the subjective response to moderate alcohol consumption; the Profile of Mood States (POMS) and the Biphasic Alcohol Effects Scale (BAES) (21)(22)(23). The POMS is a widely used questionnaire measuring subjective feelings of mood and has been often used in research interventions to investigate the influence of a treatment on mood. It was originally developed to measure mood in a clinical setting, therefore it consists of four negative mood scales (depression, fatigue, tension, anger) and just one positive mood scale (vigour). To make the POMS questionnaire more balanced, two positive mood scales from the 32-item Brunel Mood Scale are added to measure happiness and calmness (24). The BAES is a questionnaire developed specifically to measure the biphasic effects of alcohol consumption; the stimulating effects during ascending blood alcohol levels and sedative effects during descending blood alcohol levels (21,23). Previous research showed mood effects of single alcohol consumption measured by the POMS, the BAES or self-developed visual analogue scales (15,(25)(26)(27)(28)(29)(30)(31). However, there is few data on the effects of alcohol consumption with a meal and these data is inconsistent (16,17). Therefore, in this study we would like to investigate the effect of moderate alcohol consumption with a meal on the postprandial subjective response. During the postprandial state, also feelings of satiety may influence mood, but no questions on satiety are included in mood questionnaires as the POMS and BAES. However, Boelsma et al. (2010), developed a questionnaire to evaluate 'postprandial wellness' (PPW) including items on satiety, mood and gastric comfort (32) (see TNO report P5820). This questionnaire will be used to measure satiety feelings related to mood.

Physiological response
Many physiological parameters are suggested to be important factors in mood generation. For this study we will focus on physiological parameters that have been studied before in alcohol interventions in humans, because many parameters are only measurable in the brain or are very difficult to detect in peripheral blood. Therefore, the primary parameters of the physiological response are the hormones of the hypothalamic-pituitary-adrenal (HPA) axis, the Trp:LNAA ratio (reflecting serotonin response) and the ghrelin concentration in the blood.

Stress hormones
The HPA axis is a neuroendocrine system that is stimulated during stress, resulting in a fast increase in the hormones adrenocorticotropic hormone (ACTH) and cortisol in the blood (33). Previous data indicate that acute alcohol ingestion of 0.695 g/kg compared to placebo may also stimulate the HPA axis, resulting in increased ACTH and cortisol concentrations, with peak levels 10-20 min and 30 min after drinking initiation, respectively (30 TNO Protocol

Serotonin system
The effect of alcohol on mood may be mediated by different neurotransmitter systems.
The serotonin system is known to be involved in the regulation of mood and cognitive performance and previous research indicates the role of the 5-hydroxytryptamine (5-HT) receptor subtype in the effect of moderate alcohol consumption on mood (39). Brain neurotransmitter 5-HT receptor activity is shown to be influenced by the availability of the plasma precursor tryptophan (Trp) in relation to other large neutral amino acids (LNAA). The plasma Trp:LNAA ratio can be influenced by food intake. For example, a carbohydrate-rich, low-protein diet increases the ratio, resulting in higher brain 5-HT levels (40). In a study by Markus et al. (2004), it was shown that in male volunteers moderate alcohol consumption decreases the Trp:LNAA ratio after 2h (16).

Endocannabinoids
Endocannabinoids, which are conjugates of fatty acids with ethanolamides, are thought to play a role in the influence of alcohol on mood and reward. Roberto et al. (2010) showed that endocannabinoids may have a modulatory role in the effect of alcohol on the neural activity in the central amygdala (41). The acute effects of alcohol on the endocannabinoid system have been shown in cultured cells and animals, but not yet in humans (42). The results in cultured cells and animals are inconsistent. Alcohol consumption was shown to suppress the concentration of brain endocannabinoids anandamide (AEA) and 2-arachidonoylethanolamine (2-AG) (36, 43-45). However, self-administration of alcohol in rats resulted in enhanced interstitial levels of 2-AG and not AEA, measured by in vivo microdialysis, with peak 2-AG levels simultaneous to peak BAC (46). There is growing interest in a recently discovered group of fatty acid derived mediators, which are conjugates of fatty acids with serotonin, or N-acyl serotonins (47). Their function is still unknown, but they might be involved in mood. N-acyl serotonins have been shown to inhibit the activity of the enzyme fatty acid amide hydrolase in vitro, responsible for degradation of endocannabinoids (47). Therefore, in this study we are interested in the influence of alcohol on both endocannabinoids and N-acyl-serotonins and in their relation to subjective mood.

Β-endorphin
It has been suggested that β-endorphin is involved in the rewarding and reinforcing effects of alcohol. In vitro and animal studies showed that alcohol stimulates the release of β-endorphin in the brain (48). Additionally, Frias et al. (2002) showed that alcohol intoxication results in higher plasma β-endorphin levels in men and women (49). However, in a study of Dai et al. (2005) β-endorphin levels were attenuated after moderate alcohol consumption (50).

Dopamine
Another neurotransmitter involved in mood and reward is dopamine. High dopamine levels in the brain are associated with positive mood and drugs acting on the dopamine receptor are also influencing the mood state (51). In a study by Boyer et al. (2004) increased dopamine levels in the blood were found after champagne consumption, but not after still white wine consumption.
Ghrelin is an appetite-regulating hormone that has also been associated with mood, stress and anxiety. Plasma ghrelin levels were found to be increased during a stress reaction and ghrelin seems to be involved in the reward of food and alcohol (52). Food consumption results in a decline of plasma ghrelin levels, with higher calorie content P9334 | Draft | September 9 th , 2011 15 / 49

TNO Protocol
Confidential resulting in lower circulating ghrelin (53). Moderate alcohol consumption was found to attenuate ghrelin levels even more than could be expected from the caloric value, and the ghrelin levels did not return to baseline within 5h, while they returned to baseline about 3h after food consumption (36, 53-55). However, no studies have been performed on the effect of moderate alcohol consumption with a meal on ghrelin levels. After food consumption individuals may experience sleepiness. It has been suggested that CCK production, caused by presence of food (mainly lipids) in the intestine is partly responsible for the induction of sleepiness. Insulin might also be a mediating factor, however the role of insulin is less clear (1). CCK infusion in males caused sleepiness and fatigue (56, 57). In this study we will measure the effect of moderate alcohol consumption on satiety hormones, because of their potential effect on mood.

Metabolites
There is some evidence that metabolites like glucose and free fatty acids (FFA) may influence mood. Calissendorf et al. (2006) showed that low glucose levels are associated with irritability and more aggressive behaviour in frustrating situations (58 Non-invasive measures Heart rate, heart rate variability (HRV) and skin conductance level (SCL) are indices of alcohol's effect on emotional arousal. The activity of the sympathetic and parasympathetic nervous system can be estimated by heart rate variability and skin conductance level (59, 60). Previous studies showed an increased heart rate and skin conductance level after alcohol consumption (28,37,61). Heart rate variability was found to be decreased after moderate alcohol consumption (28,59,62). Though, Stritzke et al. (1995) found an attenuated skin conductance level after alcohol consumption (63). In this study, heart rate, heart rate variability and skin conductance level will be measured during baseline, ascending and descending BACs to investigate the amount of arousal during these different phases, and to determine changes in the parasympathetic and sympathetic tone.

Blood alcohol concentration
Blood alcohol concentration (BAC) will be measured in this study on the same time points as the other parameters, to be able to explain and relate the subjective and physiological responses. Objectives of the study

Primary objective
To determine whether moderate alcohol consumption with a meal in different ambiances affects postprandial mood, evaluated by subjective and physiological parameters.

Secondary objective
To investigate whether moderate alcohol consumption with a meal in different ambiances affects other physiological parameters related to mood, which are not part of the primary objective.

Primary hypothesis
It is hypothesized that moderate alcohol consumption with a meal in different ambiances changes the response of subjective and physiological parameters of mood.

Secondary hypothesis
It is hypothesized that moderate alcohol consumption with a meal in different ambiances changes the response of other physiological parameters related to mood, which are not part of the primary objective.  The study is designed as a randomized, placebo-controlled, single-blind, cross-over study. The study has a 2x2 factorial design, with treatment factors 'alcohol' (yes/no) and 'ambiance' (pleasant/unpleasant). Therefore, the subjects will visit TNO Zeist on four treatment days separated by at least 3 days, which may extend to 7 days (which is preferred). A cross-over design will be used, because large between-subject variations are expected in the subjective and physiologic responses of the primary outcomes. Subjects will be divided in 4 groups with different treatment order according to the latin square design. The test substance is a prosecco vino frizzante. The reference substance is a sparkling de-alcoholized wine. Both the sparkling white wine and de-alcoholized wine are commercially available. The de-alcoholizing process is performed by a de-alcoholizing company. During this process the wine is heated till 28 °C and applied vacuum. The study substances are selected based on a pilot study with 7 participants who scored different sparkling white wines with and without alcohol on palatability and wine taste. These wines were selected because their scores on both scales were comparable and above average (palatability: 59 for the test substance and 58 for the reference substance; wine taste: 67 for the test substance and 52 for the reference substance, with a scoring range 0-100).

Safety of handling
As the test substance and reference substance are both commercially available food products, no special precautions have to be taken in handling of the substances.

Supply to TNO
Sparkling white wine and alcohol-free sparkling white wine are both commercially available. All beverages will be purchased from one expiry date and one batch. Beverages will be supplied by a TNO employee. Products will be from single batches to limit variability between the substances.

Storage conditions
White wine and alcohol-free white wine will be stored at room temperature in a dark place. At the test days, beverages will be served cold (~5 °C) at TNO Zeist.

Chemical analyses
A few bottles of wine and alcohol-free wine out of the total batch will be stored for analysis in order to be able to perform analyses or to check alcohol percentage.

Remainder of study substances
Most of the remaining study substances will be discarded at the end of the clinical part of the study. Taking in account the possible need for additional analyses, part of the remaining study substances will be stored until the final report is accepted by the sponsor (if the nature of the substance allows this).

Description
There are two treatment factors in this study: Treatment factor 1, alcohol, consists of consumption of either: Treatment A (2x): 340 mL of sparkling white wine (3 glasses = 30 g alcohol) The meal ambiance will be manipulated by environmental factors and will be enhanced; the subjects have to watch a movie scene on a computer which is either sad or happy in the room before they start eating. The movie scenes will be from two different animation movies and will not contain content potentially shocking for some viewers.
Subjects will stay in separate rooms when they are having the intervention at TNO, to avoid social interaction with the other subjects.

Standardized meal/diet with treatment study substances
Subjects will consume a standardized meal as dinner on the treatment days. This meal will be comparable to a Dutch evening meal. Subjects have to consume the total meal and all the drinks.

Treatment code
The coding of the 2 treatment factors is as follows: Treatment factor alcohol: Treatment 'A' = sparkling white wine Treatment 'B' = alcohol-free sparkling white wine Treatment factor ambiance: Treatment '1' = pleasant meal ambiance Treatment '2' = unpleasant meal ambiance

Blinding and unblinding
The treatment factor 'alcohol' will be blinded for the subjects. They will be informed that the alcohol percentage in the wines may be different per treatment day. Furthermore, the beverage will be served in a closed cup and the serving temperature will be colder than advised (~5°C instead of ~8°C). We will check at the end of the last treatment day whether the subjects noticed the difference in alcoholic and nonalcoholic wines, by asking them the alcoholic percentage of wines of each study day (P9334 F09). The treatment factor 'ambiance' cannot be blinded, because the subjects will notice the environmental differences between the pleasant and unpleasant ambiances, but they will not be informed about it.

Confidential
Before the start of the clinical part of the study, the statistician will inform the study nurse in writing about the allocation of pre-entry number to entry number.

Supply to subjects
The meals and alcoholic drinks will be supplied to the subjects during the four treatment days, which they are obliged to finish totally. They have to consume first one glass of sparkling white wine within 5 min and afterwards they have to consume the meal and second and third glass of sparkling white wine within 15 min. So, they have 20 min in total to finish the alcoholic drinks and meal. Every subject is allocated randomly to one of the four treatment groups with a specific treatment order.

Labelling
Study substances will be labelled according to a study specific instruction as described in DHDV/ALG/711. Each bottle/box will be labelled by a pre-printed label, as follows: The TNO study number (9334) followed by a slash, followed by the 2-digit entry number, followed by a slash, followed by "treatment A" or "treatment B".

Accountability
Not applicable

Safety of treatment
The test substances (sparkling white wine and alcohol-free sparkling white wine) used in this study are commercially available and will be produced in accordance with safety and quality regulations of the commercial manufacturer. Therefore, test substances used are safe.
With an alcohol consumption of 30g (340 mL wine) the following BACs are expected for women with a body weight of 57kg -80kg: 0.38‰ -0.53‰. This has been calculated with the following formula (for alcoholic drinks with dinner in women): BAC (‰) = alcohol content (g) / (body weight (kg) * 0.5) __________________________________________________ 2 Previous research showed that simultaneous food and alcohol consumption results in attenuated BACs and that consumption of 4 alcoholic drinks (40g) with a dinner meal in men resulted in BACs not higher than 0.5‰ and (64-66).
At the end of each treatment day, the subjects BAC is measured by a breathing analyzer, and is allowed to leave TNO when her BAC has dropped below 0.2‰. This will be measured in duplo with 10 min in between.

Standardized diet
The standardized diet will consists of a macaroni dish, served by a caterer. The nutrition composition is summarized in the table on the next page.

Restrictions
Subjects will be asked to maintain their habitual diet, alcohol intake and activity pattern throughout the study. Subjects will have to refrain from alcohol for 24h prior to testing and caffeine on the afternoon of testing. Subjects will be asked to eat the same breakfast and lunch and are not allowed to exercise intensively on the 4 treatment days. Additionally, they have to refrain from eating 2h before testing.

Population base
The subjects in this study will be recruited from the pool of volunteers of TNO, location Zeist and, if needed, a recruitment office may be contacted.

Number of subjects and rationale for the number
Twenty eight women will participate in the study. The rationale for the number of subjects is based on the primary objective to determine the 'subjective response'.
Subjective response: Several scales will be used to gain knowledge on the subjective response, but the POMS is the most widely used questionnaire and also shown to be valid and reliable for measurements of mood (67). Therefore, the rationale for the number of subjects is based on the POMS. In a previous study by King et al. (1997), significant differences were picked up on the scales confusion, fatigue and vigour of the POMS. In this study 2 subgroups of 14 subjects were used, although with a slightly higher amount of alcohol, and with male subjects (25). In a study of Pasman et al. (2003), they picked up a significant difference on the scale fatigue of the POMS after consumption of two different breakfasts with 26 subjects (68). We expect to find a larger difference on the POMS scales between no alcohol or alcohol consumption with dinner, than between two breakfasts differing in macronutrient composition. Therefore, we did a power calculation based on this study. In this study a significant difference of 1.2 between the two breakfasts was shown. To reach a power of 0.8, with a significance level of 0.05 (2-sided), a difference of 1.2 (standard deviation of the difference between two values for a subject = 2.0) can be detected with 24 subjects.
To guarantee sufficient subjects more subjects will be included in case of possible dropout.

Replacement and drop-outs
A maximum of 5 subjects will be reserve until Day 01 of the study. Subjects not completing the study for any reason will be considered as drop-outs.  TNO 12. Willing to accept use of all nameless data, including publication, and the confidential use and storage of all data for at least 15 years 13. Willing to accept the disclosure of the financial benefit of participation in the study to the authorities concerned.

Inclusion criteria
* Volunteers with an age of 25-45 years, a BMI of 20-25 and alcohol consumption between 3-14 standard glasses/week are preferred for inclusion. ** With the recruitment letter, a list with accepted brands of oral contraceptive pills for participation will be added.

Exclusion criteria
Subjects with one or more of the following characteristics will be excluded from participation: 1. Participation in any clinical trial including blood sampling and/or administration of substances up to 90 days before Day 01 of this study. 2. Having a history of medical or surgical events or disease that may significantly affect the study outcome, particularly physiological disorders, or psychiatric, metabolic or endocrine disease and gastrointestinal disorders. 3. Use of medication that may affect the outcome of the study parameters (e.g. antidepressant drugs). 4. Having a family history of alcoholism 5. Having a history of alcohol or drug related problems 6. Smoking 7. Reported unexplained weight loss or gain of > 2 kg in the month prior to the prestudy screening 8. Reported slimming or medically prescribed diet 9. Reported vegan, vegetarian or macrobiotic 10. Recent blood donation (<1 month prior to the start of the study) 11. Not willing to give up blood donation during the study. 12. Pregnant (to their own knowledge) or lactating or wishing to become pregnant in the period of the study 13. Personnel of TNO Zeist, their partner and their first and second degree relatives 14. Not having a general practitioner 15. Not willing to accept information-transfer concerning participation in the study, or information regarding her health, like laboratory results, findings at anamnesis or physical examination and eventual adverse events to and from his general practitioner. Informing the subjects

Aim of the study towards the participants
The aim of the study is to investigate the effect of moderate alcohol consumption with a meal on postprandial mood. However, we keep the subjects ignorant to the aim to avoid that subject's expectations of postprandial mood effects will interfere with the pharmacologically caused postprandial mood effects. Keeping subjects ignorant is common in research with implicit measurements since the outcome of the study is easily influenced. The information subjects receive concerning the aim of the study is that we want to investigate the effect of different meal settings and alcohol on hormones and satiety. The subjects will be kept blinded for the treatment factor 'alcohol'; they will be informed that alcohol content may vary per treatment day. A blinding questionnaire at the end of the last treatment day will be used to check whether the blinding of the alcohol content was successful.

Information session and participation procedure
Candidates having informed TNO of being interested in possible study participation will be invited to come to TNO for an oral information session on the study.
The candidates will be informed verbally on the aim, the study procedures, the constraints and insurance of the study and receive a copy of the "written information for subjects" (P9334 F01).
After oral information a booklet will be handed out containing the following forms: Volunteers willing to participate will be asked to sign both informed consent forms (one for themselves and one for TNO). Thereafter subjects will be asked to complete the health and lifestyle questionnaire. The volunteers participate on a voluntary base and can withdraw at any moment of the study.
On the basis of the oral and written information, volunteers will be given two to three days to consider participation.
Each subject will be allocated to a pre-entry number consisting of the TNO study code (9334), followed by a slash ('/'), followed by a 3-digit number starting at 101.
The medical investigator will inform in writing the general practitioner of each volunteer who has signed the informed consent form on his/her application for study participation.

Study procedures and parameters
No examinations will be carried out before the volunteer concerned gave her written informed consent.
For both the pre-study period and the treatment period checklists per subject will be used.

Pre-study screen, eligibility and selection
The volunteers will have a pre-study screening before the start of the treatment period.
The pre-study screening will involve: Measurement of vital signs, venous access, height, weight and body fat percentage. The subjects also have to fill out a health and lifestyle questionnaire, the DMQ-R, STAI and DEBQ form (P9334 F02, P9334 F06, P9334 F07 and P9334 F08, respectively).
Based on the results of the pre-study screening, the medical investigator will establish the eligibility. If a volunteer is eligible the principal investigator will inform her in writing and whether she will be invited to participate in the study or is reserve or whether she is not selected. If a volunteer is not eligible the medical investigator will inform her in writing.

Anamnesis
There will be no interview (anamnesis) with the medical investigator.

Physical examination
No physical examination will be carried out.
Systolic and diastolic blood pressure and heart rate will be measured according to SOP SBJ/102, as described for screening parameters. In addition venous access will be checked.
Height will be measured according to SOP SBJ/104 (i.e. without shoes).
Whole body electrical resistance measurements will be performed by bio-impedance (according to the manual of the InBody 720). Subjects need to wear indoor clothing. Based on the data of the bio-impedance measurement, body fat percentage is measured. Body weight is also measured by the bio-impedance device (InBody 720). Data of other outcome measures of the Inbody will be not be used.

Familiarisation session
The volunteers will have a familiarisation session during the screening day. They will practice with the questionnaires to get familiar with them and the equipment for the skin conductance and heart rate variability measurements will be shown. After the screening day a macaroni dish similar to the dish served during the study will be provided to the volunteers, which they can try at home. This will reduce the possible stress present on the first treatment day.

Allocation to entry number and study treatment
After inclusion of all subjects, the statistician will randomly allocate the subjects to an entry number and a treatment group. Entry numbers will consist of the TNO study number (9334), followed by a slash ('/'), followed by a 2-digit number (01-28). The statistician will inform in writing the principal investigator or study assistant who will inform the study nurse on allocation to entry number and study treatment (see also § 8.2.3).

Assessments
The assessments to be done are summarized in the scheme below.
* Pre Study is defined as the information/screening day and is not part of the clinical part of the study. Also the familiarisation session takes place during this day. **Session 1 is defined as the first study day of the clinical part of the study. ***The visit time codes (VTCs) are based on the session number instead of study day number, because every subject will have 4 study days planned on different days. This is because subjects are only tested during the three contraceptive taking weeks, and not during the week taking no pills or hormone-free pills. The subjects can also choose to continue taking the contraceptive pill during the hormone-free week.
The first day of treatment will be specified as Session 1. There will be a wash-out period of at least 3 days, which may extend to 7 days (which is preferred). In-study subjects visit TNO in total 4 days (session 1, 2, 3, and 4) for receipt of study substances and repeated blood collection. During these test days, subjects have to refrain from caffeine during the afternoon and have to use similar breakfasts and lunches on similar time points. They have to refrain from eating 2h before visiting TNO. Subjects will come to premise at ca. 16:00h. After arrival, subjects will be prepared for blood drawing. Furthermore, three electrodes for HRV will be attached to the upper body and two electrodes for SCL will be connected to two fingers of the non-dominant hand. Both HRV and SCL will be measured continuously after attaching the electrodes until the last blood drawing. Hereafter, subjects will listen to relaxing music for about 15 min to become relaxed. Afterwards, baseline mood will be assessed by questionnaires and blood will be collected. At t=0, subjects enter a room with an unpleasant or pleasant ambiance (see 8.2.1) and get the first glass of sparkling white wine or alcohol-free sparkling white wine which they have to drink while they are watching the mood inducing scene, followed by a meal with 2 glasses of alcohol-free sparkling white wine or sparkling white wine. Subjects have to finish both the meal and wine within 20 minutes. Afterwards, subjective measures of postprandial mood will be assessed and blood will be collected repeatedly, until 150 min after the first drink. An alcohol breathing test will be performed at the end of the test day. Subjects are allowed to leave the premises if blood alcohol concentration has been measured two times <0.2‰. Subjects will stay in the room with either a pleasant or unpleasant ambiance from t=0 till they leave the premises.

Subjective response questionnaires
The subjective response will be measured by different questionnaires, filled out on the computer. The POMS is a widely used questionnaire to determine mood. It consists of 32 items, describing the mood scales depression, anger, fatigue, vigour and tension on a 5-point scale (67). Of the 5 mood scales determined by the POMS, only vigour is measuring positive mood, therefore 2 positive mood scales are added to measure happiness and calmness (based on the 32-item Brunel Mood Scale) (24) (P9334 B11). The B-BAES is a validated questionnaire to measure the stimulating and sedating effects of alcohol (23). It consists of 6 items, which will be measured by a visual analogue scale (P9334 B12  : session 1, 2, 3, and 4)  The methods used and the applicable normal values are given in P9334 B02 and P9334 B03, respectively.

Non-invasive measures
Heart rate, HRV and SCL will be measured continuously on every test day (4 days days: session 1, 2, 3, and 4). Heart rate and HRV will be calculated using continuous ECG monitoring. Three electrodes will be attached on the upper body for the ECG measurement (pre-cordial lead, sampling rate 512 Hz) . SCL will be measured using two Ag/AgCl electrodes, one positioned on the middle phalanx of the ring finger and one on the middle phalanx of the index finger of the non-dominant hand. All physiological data will be collected and stored using a multi-channel ambulatory system (Mobi-8, TMS International, The Netherlands), designed to measure different electro-physiological signals at the same time in both ambulatory and stationary conditions. The multi-channel system will be used wireless, the system can be carried in a small bag which will be attached to the subjects' belt. In this way, the subjects can move freely and will not notice much of the measurements. After storage the data will be analyzed using Matlab algorithms: Heart Rate and HRV based on R-top detection, SCL based on changes in tonic levels over the procedure. These measurements will be prepared, performed and analyzed under supervision and with assistance of Dr. Victor Kallen and Marc Grootjen from TNO Soesterberg, who have extensive experience with skin conductance, ECG measurements and the multi-channel system (69-71).

Serious Adverse Events (SAEs)
Any serious adverse event, whether or not related to the study treatment will be reported by the medical investigator immediately (within 24 hours) to the principal investigator, the sponsor or its representatives, or to the central medical committee (CCMO; www.toetsingonline.nl), the TNO-management, the general practitioner and the study subject. The exception to this rule is that serious adverse events that have already been treated by the general practitioner or medical specialist, or about which the general practitioner or medical specialist have already been informed, which situation is to be judged by the medical investigator, do not have to be reported to the general practitioner or subject. A written report on the event will be sent to the sponsor and the medical ethics committee (MEC) within 3 working days. A definition of a serious adverse event is given in P9334 B05.

Compliance with study instructions and treatment
Compliance with study instructions (e.g. pill intake, refrain from alcohol 24h before testing) will be registered on an in-study checklist (P9334 F05).

In-study samples and analyses remark
A large number of blood analyses are indicated to be performed. The actual number and precise analyses performed will be determined when the study is conducted. As stated in chapter 5 and 6 the study is hypotheses driven. Some new parameters might be interesting to be analyzed and some might not be so interesting, based on on-going research. The number of samples analyzed will be determined later. Most variables will be measured in all subjects therefore samples will be collected of all subjects at all moments a priori. However, due to budget and sensitivity of some measurements it may be possible to perform analyses with fewer samples.

Safety of subjects
The medical investigator will guard the medical safety of the subjects within the frame work of the study. She is on call 24 hours per day. During visits with blood collection, blood will be collected by an authorized person (authorized by the medical investigator), and a registered nurse or the (assistant) medical investigator will be present in the research facility.

Last assessment
At the last day of the study (or within one week after the clinical part) subjects will have an exit interview with the medical investigator only when requested by the subject and/or medical investigator.

Criteria for withdrawal or premature discontinuation
Subjects may discontinue the trial at any moment without the obligation to state the reason for discontinuation. Subjects may be withdrawn from the study by the principal investigator if they do not comply with the rules and regulations of the study. Subjects may be withdrawn from the study by the medical investigator in case of reported serious adverse events or in case of other medical/social/psychological events as evaluated by the medical investigator and discussed with the principal investigator.
In accordance to section 10, subsection 1, of the WMO, the investigator will inform the subjects and the reviewing accredited MEC if anything occurs, on the basis of which it appears that the disadvantages of participation may be significantly greater than was foreseen in the research proposal. The study will be suspended pending further review by the accredited MEC, except insofar as suspension would jeopardise the subjects' health. The principal investigator will take care that all subjects are kept informed.
Each subject who does not complete the study for any reason will have a last assessment interview, if willing to cooperate.

Payment of the subjects
• Subjects who only visit the oral information session will not be paid.
• Subjects who are screened, but who are not eligible for participation will receive €15,- (including screening). • Subjects who complete the study will receive €460,-(including screening) All amounts are excluding travel expenses; travel expenses will be calculated on reported postal code and therefore differs for each participant with a maximum of €15,per visit.
After completion of the study, subjects will fill in a complete declaration form, at the last day of the study. Subjects who dropped out during the study will be paid a sum in accordance with the procedures listed in P9334 F01. Subjects will be informed about the payment orally and in writing.
Code and handling of samples

Sample coding
In-study blood samples collected will be coded as follows: Study code (9334), followed by a slash ('/'), followed by the entry number [2 digits], followed by a slash ('/'), followed by the visit time code (VTC, P9334 B01), followed by (next line) tube code for type of matrix and volume.

Sample collection in-study
More specific information about the handling and storage of the samples, the division of the plasma and serum will be given in detail in appendix P9334 B04.

Registration of sample storage and transfer
Place of sample storage will be registered with a TNO Unique Identification number (TUI number). Sample transfer of in-study samples will be registered on forms.

Remainder samples and archive samples
Remainder of serum and plasma of the study is meant for repeated analysis in case of unexplained abnormal values and will be discarded ca. 4 weeks after reporting to the principal investigator.
Archive samples (see chapter 20) of the study period will be discarded three months after approval of the final report of the study by the sponsor and or after acceptance of scientific publication(s) concerning the study.
If necessary, archive samples will be used for analysis of markers related to the hypothesis of this study (i.e. other plasma neurotransmitters or hormones related to mood and satiety).

Documentation
The documentation of this study consists of the study protocol, correspondence, report on the results only, raw data, source documents or authenticated copies of these.
For privacy reasons, documents containing data of individual subjects will be identified only by their pre-entry or entry number. According to VOE/CLT/019 TNO has one key document in which the name of the subject is connected to pre-entry number and entry number.

Data management
The following datasets will be prepared: 1. Pre-screening parameters (age, body weight, height, BMI, body fat percentage, heart rate, diastolic blood pressure, systolic blood pressure, DMQ-R, STAI and DEBQ score.) Data at the end of each study day (session 1, 2, 3, and 4) Data at the end of each study day (session 1, 2, 3, and 4), measured continuously: 12. Non-invasive measures: heart rate, HRV and SCL Raw data of body weight, height, body fat percentage, heart rate, diastolic blood pressure and systolic blood pressure will be delivered on paper and entered in an electronic format using double data-entry. The entry of adverse events will be hand-checked by the medical investigator instead of using double data-entry.
Questionnaires (dataset 02) will be filled out on the computer and delivered in excel format.
All other raw data will be delivered in electronic format / on-line. Raw data will be registered and will be converted to SAS data. The datasets will be checked on missing and double data, on the range of the data and on correct read in and calculations of the variables. All transformations, calculations and changes (if any) in datasets will be controlled and registered.

Missing values
After completion of the clinical part of the study, the principal investigator will decide what has to be done with missing values and will inform data management. Decisions will be described in the report.

Primary hypothesis
It is hypothesized that moderate alcohol consumption with a meal in different ambiances changes the response of subjective and physiological parameters of mood.
Ho: there is no effect of alcohol consumption and ambiance on mood H1: there is an effect of alcohol consumption and ambiance on mood

Secondary hypothesis
It is hypothesized that moderate alcohol consumption with a meal in different ambiances changes the response of other physiological parameters related to mood, which are not part of the primary objective.
H0: there is no effect of alcohol consumption and ambiance on mood H1: there is an effect of alcohol consumption and ambiance on mood

Descriptive parameters
Subject characteristics will be presented descriptively.

Derived parameters
Areas under the curve (AUC) will be calculated for the parameters of the datasets 03, 04 and 05 (see par. 14.1). The AUC will be corrected for the concentration at the first measurement point. The AUC parameters will be used in the statistical analysis. Based on results and discussion with co-researchers additional AUCs may be calculated for the secondary parameters.

Analysis
The SAS statistical software package V8.2/V9.1 (SAS institute Inc., North Carolina, USA) will be used for statistical analysis.
For the subjective and physiological response over time, treatment effects will be investigated with an ANOVA taking into account the model factors: treatment 'alcohol', treatment 'ambiance', time (5 time points), the interaction between the treatments and between treatments and time. The ANOVA table is given on the next page.  The AUC measurements of the physiological response will be analysed with an ANOVA (see below).
ANOVA Based on results and discussion with co-researchers, sponsor and remarks of referee's additional statistical analysis may be performed. In all statistical tests performed, the null hypothesis (no treatment effect) will be rejected at the 0.05 level of probability. The CRO will draft a report on the clinical study and the primary outcome parameters only. This report will include aspects with regard to the used study substances, ethical aspects (list of members MEC, date of approval of the protocol and, if relevant, amendments by the MEC, details with regard to subject information and consent) and the allocation to treatment of each subject. In addition deviations from the protocol and safety data will be presented in tables and implications for the study will be briefly described. Results of the primary outcome parameters as specified in Chapter 6 of the protocol will be presented in summarizing tables. No individual data will be presented.
The final report will contain an audit certificate by the Quality Assurance Unit (QAU) of TNO Zeist and a statement on GCP compliance signed by the principal investigator. Public disclosure Policy concerning the public disclosure of the study and its results has been determined in mutual agreement between the sponsor and TNO. Neither party will unreasonably withhold the public disclosure of study results.
The following procedure is applicable: Public disclosure may include: -Registration of the study in a clinical trial register (clinicaltrials.gov); -Summarized anonymous study results issued on a TNO website, sponsors' website or an international results website (clinicaltrials.org); -Oral or poster presentation at conferences, symposia or other public meetings; -Or full publication in peer-reviewed scientific journals.
-Anonymous results of the study will be publicly disclosed either by TNO or the sponsor within 3 months after the final report has been issued.
-In case of reasonable arguments (e.g., registration procedures) a party may prolong this period up to 6 months.
-Either the sponsor or TNO is entitled to examine any form of public disclosure prior to submission. The actual start and termination dates of the study will be recorded in the final report.

Frequency of communication:
The progress of the study will be reported to the sponsor every 3-4 months at the following stages, in principle by telephone or e-mail -Approval of the protocol by the MEC -Start recruitment -Start screening -Completion recruitment and selection -Start, progress and end of clinical part of the study -Results of the study -Publication of results : + 00 31 10 40 36 100 Fax.
: 67-410035-16 The insurance covers damage with a maximum of € 450 000.-per subject, with a maximum of € 3 500 000.-for the whole study and is limited to € 5 000 000.-per insurance year. The insurance applies to the damage that becomes apparent during the study or within 4 years after the end of the study. Exclusion criteria and insurance conditions will be presented in the written information for volunteers.
The insurance certificate is specified for this study and added in appendix P9334 B08. The insurance policy is a continuous insurance; therefore deviations in study period will not affect the study insurance.
TNO has also insured the subjects for their stay, their travelling to and from TNO with a collective casualty insurance, respectively a general liability insurance. The insurance certificate is present at TNO.
Dutch law will prevail in case of legal dispute with a volunteer.
The volunteers will be informed how to handle in case of damage in the written information for volunteers (Appendix P9334 F01). Ethics and quality

General
The study will be conducted in compliance with the protocol and all amendments to the protocol. The protocol and all amendments to the protocol effecting the design, rationale or objectives of the study, or the burden of or health risks for the volunteers will only be implemented after written approval of the Medical Ethics Committee and the sponsor. All amendments will be sent to the Medical Ethics Committee for information or approval.
The study will be conducted according to:

Good Clinical Practice
TNO Zeist has been inspected by the Public Health Inspectorate for compliance with the principles of Good Clinical Practice for the conduct of clinical trials according to the EC directive 91/507/EC and the ICH Guidelines for Good Clinical Practice (CPMC)/ICH/135/95). A written statement is supplied in P9334 B06.

Quality assurance and monitoring
It is TNO policy that the Quality Assurance Unit (QAU) of TNO Zeist will audit the clinical studies. The audit certificate of the QAU will specify the dates of audits and reports to management and to the principal investigator. Members of the MEC, representatives of the sponsor and regulatory authorities may conduct inspections of the testing facility and/or the raw data and will have direct access to medical records of participants as produced and filed by the TNO Medical Investigator. To prevent interference with the study procedures, appointments for visits are requested in advance. Retention of records, samples and specimens The following documents will be retained in the archives of TNO Zeist, during 15 years after reporting of the study: 1. Master copies of the approved study protocol and final report 2. All documents containing personal data of individual trial subjects 3. Raw data (source documents or copies of these) 4. Correspondence 5. All other information related to tests and analyses conducted The following samples and specimens will be retained in appropriate facilities of TNO Zeist.
1. A representative part of the test substance will be retained for 5 years if its nature allows it. 2. Archive samples of blood will be retained for three months after the final report has been approved by the sponsor and after acceptance of scientific publication(s) concerning the study (See 12.3).  In the table below the specifics concerning blood type (plasma or serum), tube number, amount of blood and obtained on which study day, markers analysed and details about amount of dividing, handling and storage are presented.
*: remainder of serum samples will be archived. Archives and remainders will be stored at -70 ° C.

Reden van het onderzoek
In dit onderzoek willen we het effect van het gebruiken van een maaltijd in verschillende eetsituaties in combinatie met het drinken van alcohol variërend in alcoholpercentage onderzoeken.

Doel van het onderzoek
Dit onderzoek heeft tot doel het effect van een maaltijd in combinatie met verschillende hoeveelheden alcohol op hormonen en verzadiging te meten in verschillende eetsituaties.

Uitkeuring
Afhankelijk van uw wens of die van de TNO arts, heeft u een gesprek met de arts over uw huidige gezondheid en uw gezondheid tijdens het onderzoek.